Label-free detection of renal cancer

ABSTRACT

Natural and/or synthetic antibodies for specific proteins are adhered to nanoparticles. The nanoparticles are adhered to a substrate and the substrate is exposed to a sample that may contain the specific proteins. The substrates are then tested with surface enhanced Raman scattering techniques and/or localized surface plasmon resonance techniques to quantify the amount of the specific protein in the sample.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional application, which claims priority toU.S. patent application Ser. No. 13/309,852, filed Dec. 2, 2011, U.S.Provisional Patent Application No. 61/419,573, filed Dec. 3, 2010, andU.S. Provisional Patent Application No. 61/487,441 filed May 18, 2011,which are hereby incorporated by reference in their entireties.

BACKGROUND

The embodiments described herein relate generally to label-freedetection of renal cancer and, more particularly, to a method and asystem for detecting the presence of predetermined proteins via surfaceenhanced Raman scattering (SERS) or localized surface plasmon resonance.In one embodiment, the predetermined proteins are indicative of renalcancer (e.g., kidney cancer) and a patient's urine is tested for thepresence of the predetermined proteins.

Renal cancer (e.g., kidney cancer) is generally silent, frequentlyfatal, and accounts for 3% of adult malignancies. In the United Statesin 2009, more than 57,000 cases of kidney cancer were diagnosed, andalmost 13,000 deaths occurred that were attributable to this disease.Altogether, this disease represents the sixth leading cause of death dueto cancer. According to the SEER Stat Facts of the Kidney CancerHomepage of the National Cancer Institute, one in 70 adults in theUnited States will develop kidney cancer during their lifetime. For men,renal cancer is seventh among newly diagnosed cancers in 2009, ahead ofboth leukemia and pancreatic cancer. For women it is eighth in newlydiagnosed cancers, ahead of both ovarian and pancreatic cancer. Renalcancer, when symptomatically diagnosed by the classic triad of flankpain, hematuria and a palpable flank mass, has already metastasized tolymph nodes or other organs in 30-40% of patients. Renal cancer isresistant to chemotherapy, and metastatic disease portends a miserableprognosis.

There are substantial benefits to early detection. If, at diagnosis, thetumor is confined within the renal capsule, survival rates can exceed70%. Additional benefits of early detection include the opportunitiesfor laparoscopic, as opposed to open, nephrectomy and partial, asopposed to total, nephrectomy. Minimally invasive laparoscopic surgeryrather than open laparotomy enables shorter hospitalization, fasterrecovery, less pain and disability, fewer complications and lower cost.Nephron-sparing partial nephrectomy rather than total nephrectomypreserves renal mass and long-term renal function. Partial nephrectomyis associated with better long-term survival of patients with stage T1btumors than patients who undergo radical nephrectomy, suggesting thatconventional open radical nephrectomy may be considered over-treatment.The desire to preserve renal function and to minimize future chronickidney disease are compelling factors for early diagnosis of renalcancer. Identifying suitable biomarkers of kidney cancer and developmentof efficient technology to rapidly and noninvasively detect thesebiomarkers are important to disease diagnosis and documenting responseto therapy.

Kidney cancer causing carcinogens such as trichloroethylene (TCE) areubiquitously used in industrial and military applications as adegreaser. TCE is a highly toxic industrial solvent and a commoncontaminant in soil and groundwater at over 1200 sites across the UnitedStates, including hundreds of military bases—most prominently CampLejeune, N.C., the largest TCE contamination site in the country.Smoking and obesity, also common in military communities, are also riskfactors for kidney cancer. Therefore kidney cancer, itself common in thecivilian population, is an even greater threat to the health of themilitary communities, which constitute an at-risk population numberingin the millions. Kidney cancer is a deadly stealth killer, growingsilently and undetected, until so large, advanced, and usuallymetastatic, that symptoms occur. Such cancers are fatal in 95% of thevictims. In contrast, if detected early, kidney cancer can be cured inover 70% of the patients. Early detection also enables noninvasivesurgery, quick recovery, preserving kidney function, reduction in totalcost of care and minimizing disability and loss of worker productivity.There is presently no means to screen at-risk military or associatedpopulations (indeed any population) for kidney cancer.

SERS involves dramatic enhancement (up to 10¹² times or more) of theintensity of the Raman scattering from the analyte adsorbed on or inproximity to a metal surface with nanoscale roughness due primarily tothe enhanced electromagentic field. Electromagnetic enhancement dependson numerous factors such as distance of the analyte from the metalnanostructure, distance between the nanostructures in the case of dimersand aggregates, size and shape of the nanostructures, composition of themetal, and the excitation wavelength with respect to the plasmonresonance of the metal nanostructures. SERS is a powerful platform forlabel and label-free biosensing of a wide variety of biomolecules. Thereare numerous advantages to SERS based biosensors over conventionalbioassays such as ELISA, Western blotting and immuno precipitation. Themolecule-specific Raman bands (forming a molecular fingerprint) combinedwith the molecular recognition capability of the capture antibodiesimmobilized on the metal nanostructure surface enables the label-freedetection of the target biomolecules, as described herein. Furthermore,the inherently narrow Raman bands (as opposed to broad fluorescencebands of the other techniques) enable multiplexed detection of multipleanalytes in a complex mixture. Prior art SERS systems have poorsensitivity due to the poor light-metal nanostructure interaction. Inaddition, one often overlooked consideration in the design of SERSsubstrates for trace detection is the efficiency of sample collection.Known designs based on rigid substrates such as silicon, alumina, andglass resist conformal contact with the surface under investigation,making the sample collection inefficient. Accordingly, SERS substratedesigns which enable efficient guiding of the incident and scatteredphotons complemented with easy access to analytes and their selectivebinding to enable reliable real-world biological sensors are desirable.

BRIEF DESCRIPTION

In one aspect, a simple and inexpensive urine test can be performed inphysician's office for the detection of kidney cancer at a very earlystage. The test measures the quantity of two different proteins (namelyaquaporin-1 and adipophilin) present in the urine of a person. Higheramounts of these proteins are present in the urine of persons withkidney cancer compared to healthy volunteers. However, detection ofthese proteins using prior methods is expensive and time consuming.Aspects of the present disclosure enable an inexpensive technology toquickly measure the quantities of these proteins. The process is basedon surface enhanced Raman spectroscopic (light based) and/or localizedsurface plasmon resonance methods which read the fingerprint of theproteins and allow measuring the exact amount of these proteins in theurine. To achieve such detection technology a novel platform, whichenhances the fingerprint and makes it distinct from the noise associatedwith the irrelevant proteins and other molecules present in the urine,is provided.

Aspects of the disclosure can also be readily extended to the detectionof various other biomolecules in physiological liquids (e.g. blood,serum, urine). Aspects of this disclosure also provide insight into theunique properties of the nanomaterials employed in this novel sensingplatform. The sensitivity of this molecular fingerprint readingtechnology varies based on the design of the substrate comprised ofnanomaterials, which can efficiently guide the incident light tohotspots, where maximum enhancement of the signal can be attained.Aspects of the disclosure create a framework for a novel design of suchsubstrates, which are significantly better than existing designs, whichhave poor sensitivity and reproducibility.

Aquaporin-1 (AQP1) and adipophilin (ADFP) proteins in urine areexcellent candidates for the non-invasive and early detection of renalcell carcinoma (RCC). One aspect of the present disclosure demonstratesa novel plasmonic biosensor based on metal nanostructures with imprintedartificial receptors (using molecular imprinting approach) fornon-invasive and rapid screening of kidney cancer. The disclosedlabel-free assay is based on the detection of AQP1 and ADFP proteins inthe urine using localized surface plasmon resonance (LSPR) and/orsurface enhanced Raman scattering (SERS) as transduction platforms.Embodiments of the disclosure include (i) a paper based plasmonicbiosensor for sensitive and specific detection of target proteins inurine for rapid screening of kidney cancer, (ii) integrating molecularimprinting with plasmonic nanostructures to overcome issues hinderingthe progress of plasmonic biosensors to real-world application, and(iii) the application of surface force spectroscopy to probe thespecific recognition of synthetic receptors, which provides singlemolecule level understanding of the binding event.

In one aspect, the disclosure is related to the quantitative detectionof kidney cancer biomarkers (proteins aquaporin-1 and adipophilin) inurine using localized surface Plasmon resonance (LSPR) and surfaceenhanced Raman scattering (SERS) as techniques to measure thesebiomarkers. LSPR based methodology may use both glass and papersubstrates while the SERS based method may be employed using either 2Dpaper based substrates, or 3D substrates involving vertically alignednanowires or any other SERS substrates. These systems and methodsdescribed herein involve the use of either natural or syntheticantibodies for capturing the proteins from urine.

Aspects of the present disclosure enable effective and inexpensivescreening of large populations of current and former military personnel,their dependents, contractors, and civilians, including civilians nearmilitary bases, resulting in improved well-being of individualsdeveloping kidney cancer, and reduced health care costs. Aspects of theinvention further enable a rapid, simple, noninvasive, sensitive andspecific urine test for kidney cancer, which can be used to effectivelyand inexpensively screen large populations of current and formermilitary personnel, their dependents, contractors, and civilians,including civilians near military bases. Moreover, such a test can alsobe used to cost-effectively and noninvasively monitor and detectrecurrence of kidney cancer.

A SERS substrate based on filter paper adsorbed with gold nanorods,which allows conformal contact with real-world surfaces, thusdramatically enhancing the sample collection efficiency compared toconventional rigid substrates, is provided. The hierarchical fibrousstructure of paper serves as a 3D vasculature for improved uptake andtransport of the analytes to the electromagnetic hot spots in the paper.The highly efficient and cost effective SERS substrates disclosed hereinbring SERS-based trace detection closer to real-world applications.

In one aspect, a plasmonic biosensor is provided. The plasmonicbiosensor comprises a nanostructure core and functional monomerspolymerized to the nanostructure core, wherein the polymer comprises atleast one recognition cavity that is substantially complementary to atarget molecule.

In one aspect, a method of preparing a plasmonic biosensor is provided.The method comprises synthesizing a nanostructure core; immobilizing atleast one template molecule on the surface of the nanostructure core toform a template molecule-nanostructure core structure; polymerizingfunctional monomers onto the template molecule-nanostructure corestructure; and removing the template molecule to form at least onerecognition cavity upon removal of the template molecule, wherein the atleast one recognition cavity is substantially complementary to a targetmolecule.

In one aspect, a substrate comprising a plasmonic biosensor is provided.The substrate comprising a plasmonic biosensor comprises a nanostructurecore and an agent that specifically binds to a target molecule coupledto the nanostructure core, wherein the plasmonic biosensor is adsorbedto a substrate.

In one aspect, a method of preparing a substrate comprising a plasmonicbiosensor is provided. The method comprises preparing a plasmonicbiosensor by a method comprising: synthesizing a nanostructure core; andimmobilizing at least one agent on the nanostructure core, wherein theagent specifically binds to a target molecule; and adsorbing theplasmonic biosensor to a substrate.

BRIEF DESCRIPTION OF THE DRAWINGS

The embodiments described herein may be better understood by referringto the following description in conjunction with the accompanyingdrawings.

FIG. 1 is a graph of urine AQP1 concentrations in patients with andwithout renal cancer versus healthy control patients.

FIG. 2 is a perspective view of each of the steps of a fabricationroutine for producing vertically aligned ZnO nanowires with goldnanoparticle arrays, and the waveguiding of light through the resultingZnO/Au nanocob structure via multiple reflections.

FIGS. 3 a and 3 b are TEM images of (a) gold bipyramids and (b) goldnanorods.

FIG. 4 a is a schematic showing the SERS hot spot at the interstice oftwo nanopaticles.

FIG. 4 b is a schematic showing a ZnO nanorod decorated withnanoparticle dimers.

FIG. 5 is a SEM image showing the vertically aligned ZnO nanowiresdecorated with gold nanorods.

FIG. 6 is a graph of a SERS spectrum of adopophilin (1 mg/ml) on planarSERS substrate.

FIGS. 7 a-7 d are TEM images showing the extinction spectra of goldnanorods and nanobipyramids.

FIGS. 8 a and 8 b illustrate photographs of the AuNR solutions and thefilter paper before and after exposure showing the strong color change,and UV-vis extinction spectra of the AuNR solution and the AuNR loadedpaper showing the transverse and longitudinal plasmon absorption of theAuNR.

FIGS. 9 a-9 f are AFM images of a paper substrate with and withoutnanorods, an SEM image of nanorods, and an EDX image showing gold on thesurface of the paper substrate.

FIG. 10 shows the Raman spectra of filter paper collected at sixdifferent locations showing the remarkable uniformity of paper.

FIG. 11 shows the Raman spectra of pristine filter paper and filterpaper loaded with AuNRs, both treated with 1 mM of 1,4-BDT.

FIGS. 12 a and 12 b are AFM images of nanorods on a silicon surfacemodified with poly (2-vinyl pyridine).

FIGS. 13 a-13 e show Rama spectra collected from AuNR loaded paper.

FIGS. 14 a-14 c show a paper-based SERS substrate being swabbed on aglass surface to collect trace amounts and SERS spectra.

FIG. 15 is a flow chart of schematics showing the formation of themolecular imprinted plasmonic nanostructures using silanepolymerization.

FIG. 16 is a schematic showing the molecular imprinting on the surfaceof the plasmonic nanostructures bound to the surface of a substrate.

DETAILED DESCRIPTION OF THE DRAWINGS

While the making and using of various embodiments of the presentdisclosure are discussed in detail below, it should be appreciated thatthe present disclosure provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the disclosure and do not delimit the scope of thedisclosure.

To facilitate the understanding of the embodiments described herein, anumber of terms are defined below. The terms defined herein havemeanings as commonly understood by a person of ordinary skill in theareas relevant to the present disclosure. Terms such as “a,” “an,” and“the” are not intended to refer to only a singular entity, but ratherinclude the general class of which a specific example may be used forillustration. The terminology herein is used to describe specificembodiments of the disclosure, but their usage does not delimit thedisclosure, except as outlined in the claims.

While the detection of certain proteins indicative of renal cancer isdiscussed throughout, it should be appreciated that the methods andapparatuses described herein may be used in detecting other proteins orin detecting other substances in entirely different contexts. Forexample, although most prior studies clearly demonstrate that SERSsubstrates hosting closely separated metal nanostructures and/or sharptips result in large enhancements, cost and the ease and efficiency ofthe sample collection are often overlooked. In real-world applications,such as explosive detection, the efficiency of sample collection becomesa decisive factor. For example, in the case of explosives such astrinitrotoluene (TNT), which inherently have low vapor pressure (˜10ppbv at room temperature), intentional packaging further lowers theactual vapor concentration by more than an order of magnitude. Fordetection of such explosives, it is extremely important to collectparticulates (few μg), that are invariably present on the surface ofobjects exposed to the explosive. Physical swabbing, puffer systems(aerodynamic), and direct vapor sniffing are efficient methods tocollect trace amounts of analytes. In particular, swabbing the surfaceunder investigation with a soft and flexible substrate (swab) is ahighly practical and efficient method to maximize the sample collectionfrom a real-world surface. This strategy is being extensively employedfor passenger screening at airports using ion mobility spectroscopywhich involves swabbing a surface with a collector material and thenperforming ion spectroscopy on the swab and any particles on the swab.On the contrary, conventional SERS substrates based on silicon, glass,and porous alumina, which are conceived for homeland securityapplications, are not compatible with such efficient sample collectionprocess due to their non-conformal, rigid and brittle nature.

A highly efficient paper-based SERS substrate may be fabricated byloading gold nanorods (AuNR) in a commercially available laboratoryfilter paper, such as a Whatman No. 1 grade, in accordance with theprinciples described herein. The SERS substrate described herein may beused by swabbing the surface of an object suspected of exposure to ahazardous material and analyzed via SERS. The detection of less than 140pg of 1,4-benzenedithiol (1,4-BDT) residue spread over 4 cm² surface byswabbing the AuNR loaded paper on the surface is demonstrated herein.Previous attempts employing filter paper exhibited limited sensitivitypossibly due to the thin metal films (thermally evaporated or sputtered)or poor control over the size and shape of the metal nanostructuresemployed in these designs. Apart from the large enhancement, the uniformdecoration of the nanorods demonstrated herein preserves the favorableattributes such as flexibility, conformal nature, and capillarity of thepaper. These benefits and applications, as well as many others, arecontemplated as flowing from this disclosure in addition to theexemplary application of renal cancer detection.

The proteins adipophilin (ADFP) and aquaporin-1 (AQP1) in urine formexcellent candidates for the noninvasive and early detection of renalcancer carcinoma (RCC). FIG. 1 shows the AQP1 concentration in patientswith and without renal cancer, and healthy controls as determined bywestern blot analysis, expressed in arbitrary density units andnormalized to urine creatinine concentration. The plot clearly showsthat the concentrations of AQP1 in the urine of patients with eitherclear cell or papillary carcinoma were significantly greater and clearlyseparated from those in the non-RCC surgical control patients and thehealthy individuals. Very similar results were found for ADFP. Whilethese data clearly demonstrated the possibility of using these proteinsas potential biomarkers for early detection of RCC, absolutequantification of these proteins, together with the development of aninexpensive high throughput technology, is required to advance thesefindings. Conventional labeled assays such as enzyme-linkedimmunosorbent assay (ELISA) are time-consuming, expensive and requiretedious labeling procedures. These considerations clearly suggest theneed for a label-free approach for rapid and quantitative detection ofthe proteins in urine at physiologically relevant concentrations(ng/ml), as disclosed herein.

Point of care urinalysis enabling rapid screening for kidney cancer is anovel paradigm in the early detection and recurrence monitoring ofkidney cancer. A novel 3D SERS substrate comprised of vertically alignedZnO nanorods decorated with metal (e.g., a noble metal such as gold)nanoparticles with high and reproducible SERS enhancement for thequantitative detection of the protein biomarkers in urine is disclosedherein. The vertically aligned nanowires act as waveguides for theincident and Raman scattered light. As the incident light trapped insidethe nanorods traverses along the length of the nanorods, it excitessurface plasmons in the metal nanostructures adsorbed on the ZnOnanowires, thereby resulting in enhanced Raman scattering of theanalytes adsorbed on the metal nanostructures (see FIG. 2). Thedisclosed design of the SERS substrate provides distinct advantages overthe conventional 2D substrates, including, but not limited to: (i)efficient interaction of light with the metal nanoparticles decorated onthe nanowires, as the light traverses through the entire length of thenanowires, (ii) multiple reflections of the light in the waveguide willresult in enhanced probability of exciting the electromagnetic hotspots, (iii) porous nature of the SERS substrate (interstices betweenthe nanowires) provides efficient access to the analytes, (iv) highsurface area of the nanowires results in large dynamic range of thechemical and biological sensors, and (v) facile fabrication will enableefficient, robust, reliable, and cost-effective SERS substrates forhighly sensitive and selective chemical and biological sensing.

Aspects of the disclosure provide a novel, label-free assay based on thedetection of ADFP and AQP1 proteins in the urine using SERS for point ofcare and noninvasive detection of RCC. Aspects of the present disclosurealso identify characteristic Raman bands of target proteins (ADFP andAQP1) and their capture antibodies (anti-AQP1 and anti-ADFP) usingnormal Raman spectroscopy or conventional 2D SERS substrates using highconcentration of the antibody and protein. Aspects of the disclosure aredirected to fabrication of the 3D SERS substrates comprised ofvertically aligned ZnO nanowires, decoration with metal nanostructures,and immobilization of the capture anti-ADFP and anti-AQP1 on the surfaceof the metal nanostructures. The design of the SERS substrate(dimensions of the nanorods, spacing between the nanorods) may beoptimized to maximize enhancement using AQP1 in buffer as an analyte.Aspects of the present disclosure are also directed to quantitativedetection of ADFP and AQP1 in simulated (real) urine with known(unknown) concentration of AQP1 down to sub-ng/ml. The results areemployed to build the SERS intensity vs. concentration calibrationcurve. Prototype SERS-based point of service assay are tested on patientsamples to determine if quantification is identical to that of standardELISA assay.

Aspects of the disclosure provide a novel plasmonic biosensing platformbased on metal nanostructures with imprinted artificial receptors fornon-invasive and rapid screening of RCC. The label-free assay is basedon the detection of AQP1 and ADFP proteins in the urine using LSPR andSERS as transduction platforms. One aspect of the disclosure includesfabrication of gold nanostructures with artificial anti-AQP1 andanti-ADFP on the surface. Accomplishing this aim involves synthesis ofgold nanostructures (e.g. nanorods and bipyramids), immobilization ofthe proteins (i.e. templates) and polymerization and crosslinking of thefunctional monomer around the template followed by the removal of thetemplates. Another aspect of the disclosure includes quantitativeunderstanding of the specific binding efficacy of the syntheticreceptors using surface force spectroscopy. Atomic force microscopy(AFM) based surface force spectroscopic measurements is employed toprobe the specific interactions between the synthetic anti-AQP1 andtarget biomolecules by functionalized AFM tips. The measurements willprovide a valuable insight into various parameters (e.g. choice ofmonomers, thickness of the polymer layer, crosslinking density), whichare critical for the design of the synthetic receptors. Naturalmonoclonal antibodies against the target proteins will be employed ascontrols to compare the binding efficiency of the synthetic receptors.Another aspect of the disclosure includes plasmonic biosensing of thetarget biomolecules at physiologically relevant conditions using a paperbased platform. Metal nanostructures with synthetic receptors on thesurface will be immobilized on paper substrates to perform LSPR and SERSmeasurements. Statistical data analysis is performed to demonstrate thedetection of target biomolecules at physiologically relevantconcentrations (sub-ng/ml) and compared to standard enzyme-linkedimmunosorbent assay (ELISA).

Surface plasmon involves the collective coherent oscillation of theconductive electrons at the interface of metal and dielectric materials.Based on the sensitivity of the surface plasmon resonance to thedielectric ambient and the enhancement of electromagnetic field inproximity to metal nanostructures, two important classes of plasmonicbiosensors (LSPR and SERS) are being actively investigated. LSPR- andSERS-based biosensing platforms have enormous potential to providehighly sensitive, cost-effective and point-of-care diagnostic tools.However, the state of the art approach (i.e. using natural antibodies ascapture agents) presents several impediments in translation of thesesensors to the real-world. Aspects of the present disclosure addressthis issue by designing and synthesis of plasmonic nanostructures withbuilt-in synthetic receptors on the surface using a molecular imprintingapproach. These novel plasmonic nanostructures with biofunctionalityenable highly sensitive and specific biosensors for rapid screening ofRCC. Apart from their impact in diagnostics, the plasmonicnanostructures disclosed herein with synthetic antibodies can also haveapplications in photo-thermal imaging (targeted accumulation),photo-thermal therapy, targeted drug-delivery and even neutralization ofother biomolecules.

Synthesis of Metal Nanostructures

Owing to the sharp corners (electromagnetic hot spots) and faciletunability of the plasmon resonance by controlling the aspect ratio,gold nanorods and gold nanobipyramids are used for the fabrication ofSERS substrates. The plasmon resonance wavelength half-way between theRaman source laser and Stokes-shifted Raman scattered light forms theideal condition for maximum SERS enhancement. Gold nanorods andnanobipyramids are prepared using seed mediated approach, as describedin more detail herein. FIGS. 3 a and 3 b show the TEM images of goldnanorods and nanobipyramids.

Efficiency of the SERS substrates can be enhanced by using dimers ofgold nanorods and gold bipyramids instead of individual nanostructures(FIGS. 4 a and 4 b). The electromagnetic interaction (electromagneticcoupling) between metal nanostructures greatly increases the SERSintensity. In view of the large SERS enhancements from dimers andmulti-particle aggregates, significant efforts have been made tochemically synthesize dimers and trimers of metal nanoparticles. Afacile strategy, which involves a careful control of the amount ofchloride added during synthesis of metal nanostructures to enabledimerization of metal nanostructures may be employed to form dimers ofnanoparticles followed by their adsorption onto ZnO nanorods. Oneconsideration in using dimers is the significant dependence of theelectromagnetic enhancement on the polarization of light with respect todimer axis. The SERS enhancement is maximized when the polarization isparallel to the dimer axis. The assembly of the dimers is tuned toorient the dimer axis parallel to the substrate by manipulating thedrying (directional drying by tilting the substrate) of the nanoparticlesolution on the vertically aligned nanowires (see FIG. 4 b).

Synthesis of ZnO/Metal Nanostructure Hybrids

The ZnO nanowires may be grown using a solution approach in which ZnOseed layer (or gold with titanium adhesion layer) is deposited usingmagnetron sputtering or by spin casting zinc acetate solution on asilicon substrate (which can be replaced with a polymer or papersubstrate at later stages). For the fabrication of vertically alignedZnO nanowires, the substrates are exposed to a solution of zinc nitratehexahydrate and hexamethylenetetramin Substrates are subsequently rinsedwith a mixture of ethanol and water and oven dried to result inuniformly oriented ZnO nanowires with a diameter of 100-300 nm and alength of 5-10 μm (see FIG. 5). The surface of the vertically alignedZnO nanowires is modified with a monolayer of weak cationicpolyelectrolyte, poly (2-vinyl pyridine) (P2VP) by adsorption fromethanol solution followed by extensive rinsing. One challenge forreal-world application of the SERS as a biosensing platform is theability to reliably and repeatedly quantify the analyte. Several factorswhich determine the uniformity of the envisioned SERS substrates are:(i) areal density of the vertically aligned ZnO nanowires, (ii) densityof the nanoparticle or their aggregates decorating the ZnO nanowires,(iii) porosity (which determines the access to analytes) of the SERSsubstrate, and (iv) uniformity of the selective coating preferentiallyabsorbing (or adsorbing) the analytes. Fabrication conditionsinfluencing these various features may be identified and controlled toachieve a highly reproducible SERS enhancement within the substrate andfrom substrate to substrate.

Gold nanorods and gold nanobipyramids and their dimers are immobilizedon the surface of the ZnO nanowires modified with P2VP, resulting in thevertically aligned hybrid nanostructures (see FIGS. 2 and 4). The nextstep involves the immobilization of the capture antibodies (anti-ADFPand anti-AQP1) on the gold nanostructures for selective binding of thetarget proteins. One consideration for the immobilization of the captureantibodies is retaining their ability to selectively capture targetproteins. The capture antibodies are immobilized through protein A,which has specific affinity to gold and Fc fragment of the antibody. Theimmobilization protocol will involve the immobilization of protein A onthe surface of metal nanostructures followed by immobilization ofcapture antibodies. Immobilization of capture antibodies and thefunctionality of the capture antibodies are verified by usingfluorescently labeled target protein and monitoring the binding of theseproteins to the capture biomolecules from buffered solution.

SERS Measurements and Statistical Data Analysis

Characteristic Raman bands of AQP1, ADFP and the correspondingantibodies are identified using bulk Raman measurements or usingconventional 2D SERS substrates and a high concentration of thebiomolecules (1 mg/ml). The proteins are immobilized on metalnanostructure using histidine tag (hexa-histidine residues), whichenables controlled appendage of the protein to the metal nanostructurewith high regiospecificity. The imidazole ring of hisitidine adsorbswith flat-on geometry to the gold nanoparticle with high adsorptionenergy (˜35 Kcal/mol). FIG. 6 shows the Raman spectrum of the his-taggedADFP obtained by depositing the protein on planar SERS substrate. Ramanspectrum clearly shows the strong characteristic bands of the protein.Following the identification of the Raman bands, the 3D substrates willbe employed for detection and quantification of the target protein attrace levels (1 μg/ml-0.1 ng/ml). The hybrid nanostructures with captureantibodies will be exposed to various concentrations of the targetproteins in buffered environment or in simulated urine followed bythorough rinsing to remove the non-specifically adsorbed protein. SERSspectra may be collected using a confocal Raman spectrometer mounted ona Leica microscope equipped with 514.5 and 785 nm lasers or a hand-heldspectrometer. For 785 nm wavelength laser, which works well for goldnanostructures and biomolecules, the focal volume (and spot diameter) ofthe laser focused with 20× and 50× objectives is 32.3 fl (1.20 μm) and2.61 fl (0.64 μm), respectively. The small spot size will enable probingindividual ZnO nanowires and their ensemble. Furthermore, SERS and highresolution SEM can be obtained from the same location (even samenanowire) using external markers, which enable the correlation of theobserved SERS intensity and the hybrid ZnO/metal nanostructure complex.This system and method of collecting SERS spectra data is merelyexemplary, and it is contemplated that various other SERS spectra datacollection methods and systems may be utilized within the scope of theclaims.

The ability to distinguish similarly structured but different chemicalspecies from a complex mixture of biological species using a spectralmethod requires a sufficient variability in the spectra of the species,and this variability should be greater than the variance due toinconsistencies that may arise from small differences in samplepreparation. For moderate detection levels (concentration>1 μg/ml), SERSalready provides distinct spectral differences due to the strong Ramanbands, which are enhanced 105-109 times compared to normal Ramanscattering. However, at trace level detection (concentration<100 ng/ml),the spectral differences can be subtle for distinguishing the chemicalspecies. To achieve the trace level analysis, multivariable statisticalmeans, such as principal component analysis (PCA) via intrinsic Ramanspectra of the analyte of interest, may be employed. Specifically,linear multivariable models of SERS spectra data sets may be built byestablishing principal component vectors (PCs), which will provide thestatistically most significant variations in the data sets, and reducethe dimensionality of the sample matrix. This approach involvesassigning a score for the PCs of each spectrum collected followed byplotting the spectrum as a single data point in a two-dimensional plot.The plot will reveal clusters of similar spectra, thus individualbiological species (analyte and interfering molecules) can be classifiedand differentiated for even closely related ones.

Example Comparison of 3D SERS Assay with Standard ELISA Assay

Two patient cohorts providing banked urine samples are provided. Thefirst consists of 67 patients with histology-proven clear cell andpapillary kidney cancer. Tumor size ranges from 0.6 to 18 cm with mostbeing in the T1a stage of up to 4 cm. The second cohort consists of 54control patients having surgery for non-kidney related issues that arematched by age, sex, weight, and smoking tendency to the kidney cancerpatients. Obesity and smoking, both prevalent in the military, are riskfactors for kidney cancer. AQP1 and ADFP levels are quantified in thereal urine samples using the SERS approach and standard ELISA assay. Thecorrelation between the concentrations of the proteins obtained fromthese independent techniques are analyzed using partial least squares(PLS) method. A standard commercial PLS package (e.g., PLS Toolbox) isemployed for performing the statistical analysis to compare SERS assaywith the gold standard (ELISA).

Nanoparticle Construction

Owing to their high refractive index sensitivity and facile tunabilityof the plasmon bands, gold nanorods (˜250 nm/RIU) and goldnanobipyramids (˜600 nm/RIU) are used as plamonic nanostructures. FIGS.7 a-7 d show the TEM images and the extinction spectra of gold nanorodsand nanobipyramids.

Example Construction of Gold Nanorod on Paper

According to one aspect of this disclosure, gold nanorods weresynthesized using a seed-mediated approach using cetyltrimethylammoniumbromide (CTAB) as a capping agent, as described in more detail herein.The nanorods were found to be ˜80 nm long and ˜20 nm in diameter, makingthe aspect ratio to be nearly four. Exposing the filter paper toCTAB-capped gold nanorod solution resulted in uniform adsorption of thenanorods on the surface of the paper and a color change from white topurple (FIG. 8 a). A significant decrease in the intensity of the purplecolor of the AuNR solution was observed after removing the paper fromthe solution (see FIG. 8 a), which corresponded to nearly 50% decreasein extinction intensity. This significant change in the intensity of thecolor of the solution following the filter paper exposure is inaccordance with the high density of nanorods on the surface of the paperand deep purple color of the paper. UV-vis extinction spectra of theAuNR solution showed the two characteristic peaks at ˜530 nm and 650 nmcorresponding to the transverse and longitudinal plasmon resonances ofthe nanorods, respectively (see FIG. 8 b). AuNR loaded paper exhibitedsimilar extinction spectrum with both transverse and longitudinalplasmon slightly blue shifted compared to the solution. The blue shiftobserved can be attributed to the change in the dielectric ambient (fromwater to air+substrate) with an effective decrease in the refractiveindex. The blue shift of the longitudinal plasmon peak (34 nm) was foundto be slightly higher compared to the transverse band (12 nm), which canbe attributed to the higher sensitivity of the longitudinal plasmonresonance to the changes in the dielectric ambient compared to thetransverse band.

Cellulose is biodegradable, renewable, and abundant in nature thuscellulose (or paper) based products can be inexpensively produced andrecycled. Due to numerous advantages such as significant reduction incost, high specific surface area, excellent wicking properties, andcompatibility with conventional printing approaches (enabling multiplexdetection and easy disposability) paper is gaining increased attentionas a substrate in diagnostic and tissue engineering applications.According to one aspect of this disclosure, paper may be constructedfrom cellulose fibers. Alternatively, or additionally, paper, or afibrous mat, may be constructed from other materials, including wovenand/or non-woven fibers (e.g., polymer fibers) and used as a substrate.FIG. 9 a shows the hierarchical fibrous morphology of the filter paperwith cellulose nanofibers braided into microfibers (average diameter of˜0.4 μm). The RMS surface roughness of the paper was found to be 72 nmover 5×5 μm² area, which indicates the large surface area of the papersubstrates. Raman spectra obtained from six different areas of thepristine filter paper with 1 inch diameter exhibited excellentcompositional (spectral) homogeneity, which may be important for itsapplication as a SERS substrate (see FIG. 10). It is well known thatuniform and high density adsorption of CTAB (cationic surfactant) cappedAuNR to polymer surfaces is a significant challenge. It may be observedthat once the AuNR loaded paper was dried, even under vigorous rinsingwith water or alcohol, no noticeable change in the AuNR density wasobserved, suggesting the stability of these substrates for deployment inliquid environments. Cellulose has a large number of hydroxyl groups,which are accessible for attaching positively charged species. Theuniform, irreversible, and high density adsorption of the AuNR ispossibly due to the electrostatic interaction between the positivecharged nanorods and the filter paper.

AFM imaging revealed a uniform and dense adsorption of nanorods on thesurface of the paper without any signs of large scale aggregation of thenanorods (FIG. 9 b). Higher magnification AFM images show the nanorodsdecorating the fibers of the paper and a partial local alignment of thenanorods along the nanofibers (FIGS. 9 c, 9 d). From numerous AFM imagescollected at different areas of the substrate, the number density of thenanorods was found to be 98±22/μm². High magnification SEM image showsthe uniformly adsorbed gold nanorods on the paper (FIG. 9 e). Energydispersive X-ray spectra (EDX) confirmed the presence of gold on thesurface apart from the carbon and oxygen rich cellulose fibers (see FIG.9 f).

1,4-Benzenedithiol (1,4-BDT) is widely employed as a model analyte forSERS owing to its ability to readily adsorb on gold or silver particlesand its distinct Raman fingerprint. The Raman spectrum of 1,4-BDT inneat solid state exhibits strong bands at 740, 1058, 1093, 1186, and1573 cm-1. Three prominent bands: 1058 cm-1 due to the combination ofthe phenyl ring breathing mode, CH in-plane bending, and CS stretching,1181 cm-1 due to CH bending, and 1562 cm-1 due to phenyl ring stretchingare commonly employed as characteristic peaks for evaluating theperformance of SERS substrates. The 1058 cm-1 band was utilized to testthe performance of the SERS substrate in detecting trace amounts of1,4-BDT in ethanol. The pristine SERS substrate (AuNR loaded paper) doesnot show any peak in this region (see FIG. 11).

As a planar rigid substrate for comparison, a highly dense (numberdensity: 220±14/μm²) layer of gold nanorods bound to silicon substratemodified with poly(2-vinyl pyridine) was employed (FIG. 12). FIG. 13shows the Raman spectra obtained from the planar AuNR control sample andAuNR loaded paper both exposed to 1 mM of 1,4-BDT, followed by rinsingwith ethanol. While the Raman spectra obtained from the AuNRs on siliconsubstrate exhibits weak Raman bands of 1,4-BDT, the AuNR loaded paperexhibited much stronger (˜250 times) Raman bands. These Raman spectraobtained from the SERS substrates exhibited small shifts in frequency ofthe vibrational bands compared to the bulk 1,4-BDT (i.e. 1185 cm-1 forbulk BDT, and 1180 cm-1 for SERS substrate), possibly due to theorientation change of 1,4-BDT molecules adsorbed on to the AuNR.

To investigate the trace detection ability of the paper based SERSsubstrate, Raman spectra were collected from substrates exposed to1,4-BDT down to concentrations of 0.1 nM. All the characteristic bandsof the 1,4-BDT exhibited a monotonous decrease in intensity withdecreasing concentration (FIG. 13 b). FIG. 13 c shows the higherresolution spectra of the smoothed 1058 cm-1 band, which is clearlydistinguishable (signal to noise ratio of 3) down to a concentration of0.1 nM (17 ppt) (see FIG. 14, illustrating higher resolution spectra).Semi-log plot of the concentration vs. 1058 cm-1 peak intensity shows amonotonic increase in the Raman intensity with increasing concentrationof the analyte (FIG. 13 d). Data from FIG. 13 d can be plotted as theinverse of the fractional coverage (taken as a ratio of intensity,Imax/I) with respect to the inverse of the concentration (1/c), whichexhibits a linear relationship, reflecting the expected Langmuiradsorption isotherm of 1,4-BDT to gold nanorods (see FIG. 13 e).

The following expression was used to calculate the enhancement factor(EF) of SERS substrate at 1058 cm-1 band:

EF=I _(SERS) ×N _(bulk) /I _(bulk) ×N _(SERS)  (1)

where ISERS (NSERS) and Ibulk (Nbulk) are the intensities (the number of1,4-BDT molecules probed) for the SERS and bulk spectra, respectively.40 NSERS was estimated by assuming a complete monolayer of 1,4-BDT onthe nanorods for SERS substrates exposed to 1 mM concentration, whichensures that the enhancement factor is not overestimated. Based onnumerous AFM images, the areal coverage of the AuNR was estimated to be˜23% and NSERS was calculated to be 4.9×105 molecules. Ibulk and Nbulkwere determined from the Raman spectra of a 0.1 M of 1,4-BDT in 12 MNaOH(aq) (see experimental for details). Using the SERS intensity of the1058 cm-1 band, the enhancement factor was calculated to be ˜5×106. Theenhancement factor observed here is high considering the absence of anyresonance contribution, the use of gold nanostructures as opposed tosilver nanostructures, which result in higher enhancement at the expenseof poor long-term stability, absence of any intentionally formed hotspots (dimers or controlled aggregates) and the simplicity of thefabrication approach.

One of the distinct advantages of the paper based SERS substrate is theability to collect trace amount of analytes from real-world surfaces byswabbing across the surface. This unique ability of the paper substratesis demonstrated by swabbing a slightly wetted (in ethanol) paper onsurface of a glass with trace quantities of analyte deposited on thesurface (see FIG. 14 a). FIG. 14 b shows the Raman spectra (averagedover 6 different spots) obtained by swabbing the paper across thesurface with different amounts of analyte. Again, the strongest Ramanband at 1058 cm-1 was used to evaluate the efficiency of the SERS swab.It can be seen that the Raman bands of 1,4-BDT can be clearlydistinguished down to 140 pg on the surface (FIG. 14 c). Consideringthat the swabbing of the surface results only a fraction of the analyteto be absorbed into the paper, a detection limit on the order of fewtens of picograms on the surface is truly remarkable.

Envisioned and designed as an end-user level SERS substrate, properhandling of paper SERS substrate becomes an important issue as theycontain metal nanostructures, which could be potentially harmful tohumans and the environment. Toxicity of gold nanoparticles is stilldebated even though many reports indicate that gold nanoroparticles areessentially nontoxic. In a recent perspective, it was suggested that thetoxicity and cell uptake of gold nanorods can be controlled to a pointthat they would not pose a serious harm by functionalizing the surfaceof gold nanorods with biocompatible ligands. A similar approach (i.e.,tailoring the surface chemistry of the metal nanostructures) may beemployed to make the paper SERS substrates bio-friendly.

Experiment: Gold Nanorod on Paper

Gold nanorods had been synthesized using a seed-mediated approach. 25,26Seed solution was prepared by adding 1 mL of an ice-cold solution of 10mM sodium borohydride into magnetically stirred 10 mL of 0.1 Mcetyltrimethylammonium bromide (CTAB) and 2.5×10-4 M HAuCl4(aq) solutionat room temperature. The color of the seed solution changed from yellowto brown. Growth solution was prepared by mixing 95 ml of 0.1 M CTAB, 1ml of 10 mM silver nitrate, 5 ml of 10 mM HAuCl4, and 0.55 ml of 0.1 Mascorbic acid in the same order. The solution was homogenized by gentlestirring. To resulting colorless solution, 0.12 ml of freshly preparedseed solution was added and set aside in dark for 14 hours. The solutionturned from colorless to violet brown with most of the color changehappening in the first hour. Prior to use, the gold nanorod solution wascentrifuged at 13,000 rpm for 10 mM to remove excess CTAB andredispersed in nanopure water (18.2 MΩ-cm). The procedure was repeatedtwice. AuNRs are loaded in a laboratory filter paper (Whatman No. 1grade) by immersing a 1 cm² paper in 2.5 mL of AuNR solution for twodays. Upon removing from the solution, the paper was gently rinsed withnanopure water and then blow-dried under a stream of dry nitrogen.Planar silicon substrates for comparison were fabricated by modifyingthe silicon substrate with poly(2-vinyl pyridine) (P2VP) by exposing thepiranha cleaned silicon surface to 4% P2VP solution in ethanol. Afterrinsing the silicon substrate with ethanol it was exposed to goldnanorod solution to enable adsorption of the gold nanorods. Finally, thesubstrate was rinsed with water to remove the loosely bound nanorodsleaving a highly dense layer of nanorods on the surface.

For the dipping test, the performance of detecting a trace amount of1,4-BDT was evaluated by dipping the SERS substrate in variousconcentrations of 1,4-BDT in ethanol for 20 minutes, followed by lightrinsing with ethanol and drying with compressed nitrogen gas before theRaman measurements. Six Raman scans were performed for each substratewith each scan representing a different spot within the same substrate.The Raman data were averaged and normalized against 1058 cm-1 band.

For the swabbing test, 100 μL of 1 μM to 1 nM 1,4-BDT (corresponding toapprox. 14 μg to 14 pg) in ethanol was pipetted on the surface of aglass slide, which immediately spread over 4 cm2 area. Evaporation ofethanol left residue of 1,4-BDT. A drop of ethanol was placed on a 0.5×1cm SERS substrate to wet, and then swabbed the surface of the glassslide to pick up the residue of 1,4-BDT. Raman spectra of the swabbedSERS substrate were collected on six different spots. The Raman spectrawere averaged and normalized against 1058 cm-1 band.

Raman spectra were measured using a Renishaw inVia confocal Ramanspectrometer mounted on a Leica microscope with 20× objective (NA=0.40)in the range of 100-3200 cm-1 with one accumulation and 10 s exposuretime. A 785 nm wavelength diode laser (0.5 mW) coupled to a holographicnotch filter with a grating of 1200 lines mm-1 was used to excite thesample. The following expression was used to approximate the laser spotsize (1.2 μm in diameter)

$\begin{matrix}{w_{0} = \frac{0.61 \times \lambda}{NA}} & (2)\end{matrix}$

where w₀ is the minimum waist diameter for a laser beam of wavelengthfocused by a objective with a numerical aperture NA. The focal volume(t) was approximated from the following expressions

$\begin{matrix}{\tau = {( \frac{\pi}{2} )^{1.5}w_{0}^{2}z_{0}}} & (3) \\{z_{0} = \frac{2\pi \; w_{0}^{2}}{\lambda}} & (4)\end{matrix}$

where z₀ is the focal depth.

SEM images were obtained using a FEI Nova 2300 Field Emission SEM at anaccelerating voltage of 10 kV. AFM images were obtained using Dimension3000 (Digital instruments) AFM in light tapping mode. UV-vis spectrawere measured using a CRAIC microspectrophotometer (QDI 302) coupled toa Leica optical microscope (DM 4000M) and a Shimadzu UV-1800 UV-visspectrophotometer.

Surface Force Spectroscopic Measurements

In order to probe the specific recognition capabilities of the molecularimprinted nanostructures, AFM based force spectroscopy measurements areperformed. All the force measurements are performed in controlledaqueous environment (phosphate buffer, pH 7.4) using a standard fluidcell, eliminating the contribution of capillary forces in the observedadhesion force. Commercially available microfabricated cantilevers withintegrated probes with a nominal radius of less than 10 nm and springconstant of 0.05-0.5 N/m may be employed. The shape of the AFM tips aredetermined by scanning gold nanoparticle (diameter of 5 nm) standardsamples. Cantilever spring constants may be obtained using thermaltuning or spring-on-spring measurements. In the initial stages, surfaceforce spectroscopic (SFS) measurements are performed on planar siliconsubstrates with imprinted polysiloxane film.

Numerous functionalization strategies have been developed to bind theprotein molecules to the surface of the AFM tip, each with its own setof advantages and shortcomings. The his-tag on the protein may beemployed to absorb the protein to the gold coated AFM tip. His-tagenables the region specific binding of the protein to the surface gold.The his-tag on the protein may space the AQP1 from the surface of thetip to preserve the natural conformation, thus it significantly reducesthe non-specific adsorption onto the tip and provides necessaryorientation freedom.

LSPR and SERS Measurements

Paper based substrates may be employed for performing the LSPR and SERSmeasurements on the molecular imprinted metal nanostructures. Papersubstrates are exposed to the molecular imprinted plasmonicnanostructure solution followed by extensive rinsing and drying. AFM andSEM are employed to quantitatively probe the density of thenanostructures and uniformity of the substrate over large areas.Subsequently, LSPR/SERS biosensing measurements are performed byexposing the paper to simulated physiological liquid with variousconcentrations of the target biomolecules (discussed below). Finally,AQP1 and ADFP levels are quantified in the real urine samples using theSERS approach and standard ELISA assay. The correlation between theconcentrations of the proteins obtained from these independenttechniques may be analyzed using partial least squares (PLS) method.Standard commercial PLS package (PLS Toolbox) will be employed forperforming the statistical analysis to compare SERS assay with the goldstandard (ELISA).

Artificial Antibodies on Gold Nanostructures

Two designs for integrating plasmonic biosensing (LSPR and SERS basedapproaches) with synthetic receptors using molecular imprintingdisclosed herein involve surface imprinting of the artificial receptorsonto the metal nanostructures. As disclosed herein, surface imprintingas opposed to the bulk imprinting offers distinct advantages in that thecapture sites are readily available to the analyte moleculessignificantly reducing the sensor response time. Furthermore,considering the surface sensitive nature of the transduction chosen inthis study (LSPR and SERS), bulk imprinting may not provide anyadditional advantages such as higher sensitivity or larger dynamicrange. Two different surface imprinting strategies are described herein.

Molecular Imprinted Nanostructures

In one approach, silane polymerization is exploited to achieve molecularimprinted artificial antibodies on the surface metal nanostructures (seeFIG. 15). The silane polymerization will be performed by sol-gelmethods, where the polysiloxane networks are formed by siloxane bondsbetween silanol groups. The surface of the silica shells of AuNP@SiO2(i.e., the silica-coated gold nanostructures) may be modified byintroducing amine functionality using 3-aminopropyltrimethoxysilane(APTMS). Aldehyde functionality may then be introduced usingglutaraldehyde where the amine groups on the silica surface areconverted to aldehyde groups. Hemoglobin (or His-tagged AQP1 andhis-tagged ADFP) may be bound to the surface of the AuNP@SiO2 using thealdehyde groups on the surface of these particles, forming imine bonds.Following the immobilization of the proteins on the surface of themetal-silica nanostructures, organic silane monomers, APTMS andpropyltrimethoxysilane (PTMS) may be polymerized onto thehemoglobin-silica (or AQP-1-silica) surface at room temperature underbuffered conditions to prevent the denaturing of the protein molecules.Concentration of PTMS below 60% facilitates the easy removal of thetemplate molecules after the silane polymerization. The template maythen be removed by washing with organic acid or acidic reagents such asoxalic acid. Oxalic acid breaks the imine bond between the silicasurface and the protein, thus removing the proteins from the surface.The removal of the template leaves recognition cavities, which act asspecific recognition sites for the rebinding of the protein. Theschematic of the imprinting of protein onto silica surface is shown inFIG. 8. One of the challenges for reliable biosensing is overcomingnon-specific binding of the biomolecules to the active sensing layer.The possibility of non-specific binding even in the case of themolecularly imprinted nanostructures is contemplated. The non-specificinteractions may be too strong to overcome even with thorough washingwith buffer solution. To overcome this issue, polyethylene glycol (PEG)may be used as a passivating layer. This can be achived in two ways: (i)polysiloxane network will be formed using sliane monomers functionalizedwith PEG side chains or (ii) grafting PEG chains to polysioxane networkbefore removal of the template molecules. PEG has been demonstrated tobe efficient in preventing non-specific binding of the biomolecule andenhance the sensitivity of plasmonic sensors.

Surface Imprinting on Immobilized Particles

In an alternate approach, surface molecular imprinting will be achievedusing soft substrates, which can make conformal contact with metalnanostructures immobilized on the surface (see FIG. 16). The surfaceimprinting process will start by immobilizing hemoglobin onto oxidizedpolydimethyl siloxane (PDMS) substrates. Amination of the PDMSsubstrates will be performed by exposing the substrates to APTMS.Aldehyde groups for binding the hemoglobin to the surface of PDMS-NH2will be formed by reacting with glutaraldehyde. Hemoglobin will be boundto the surface of the PDMS-NH2 with aldehyde groups forming imine bondsas described above. Successful immobilization of the hemoglobin will beverified by fluorescence (using labeled protein) and AFM. Following theimmobilization of the protein on PDMS surface, a functional monomer suchas metahcrylic acid (MAA), which possesses carboxylic groups, will beapplied to the PDMS with hemoglobin. Following the assembly of themonomer around the protein by non-covalent interactions such as hydrogenbonding, excess monomer will be removed by gently washing in alcohol.Subsequently, the surface will be inked in crosslinker (divinylbenzene), radical initiator (benzylperoxide) and an anchoring monomer(triethoxyvinylsilane(TVES)) causing the polymerization of thefunctional monomer around the hemoglobin molecules. In the next step,PDMS substrate will be brought in conformal contact with the gold/silicacore-shell nanoparticles immobilized on the surface of a substrate,followed by peeling the PDMS off to leave the molecular imprints on thesurface of the nanostructures on the surface. The AuNP@SiO2 or Au NPwill be immobilized on the surface of silicon substrate usingpoly(2-vinylpyridine) (P2VP) as an anchoring layer. Silicon surface willbe modified with P2VP by adsorption from dilute solutions or by spincoating approach. The TVES layer will act as an anchoring layer forbinding the imprinted receptors to the silica coated nanoparticles. Inan alternate approach, polyglycidyl methacrylate (PGMA) may be employedas an anchoring layer for immobilization of the polymerized MAAreceptors directly onto the gold nanoparticle immobilized onto thesubstrate. Owing to the sticky epoxy groups, PGMA exhibits excellentbinding to a wide variety of organic and inorganic species.

Immobilizing Natural Antibodies on Gold Nanostructures

One of the considerations for the immobilization of the captureantibodies is retaining their ability to selectively capture targetproteins. The capture antibodies may be immobilized through protein A,which has specific affinity to gold and Fc fragment of the antibody. Theimmobilization protocol may involve the immobilization of protein A onthe surface of metal nanostructures followed by immobilization ofcapture antibodies. Immobilization of capture antibodies and thefunctionality of the capture antibodies may be verified by usingfluorescently labeled target protein and monitoring the binding of theseproteins to the capture biomolecules from buffered solution.

It will be understood that the particular embodiments described hereinare shown by way of illustration and not as limitations of thedisclosure. The principal features of this disclosure may be employed invarious embodiments without departing from the scope of the disclosure.Those of ordinary skill in the art will recognize numerous equivalentsto the specific procedures described herein. Such equivalents areconsidered to be within the scope of this disclosure and are covered bythe claims.

An efficient SERS substrate based on common filter paper filled withgold nanorods is described herein, which exhibited more than two ordersof magnitude higher SERS enhancement compared to the silicon based SERSsubstrate. Numerous favorable traits of the paper such as flexibility,conformability, efficient uptake, and transport of the analytes fromliquid and solid media to the surface of metal nanostructures due tohierarchical vasculature and high specific surface area make the paperbased SERS substrates demonstrated here an excellent candidate for tracechemical and biological detection. The paper based SERS substrates alsooffer cost-effective platform for SERS detection and opens up a newvenue for other biological and chemical detection. The processdemonstrated here can be easily scaled up for batch fabrication of SERSswabs. Furthermore, the paper-based SERS substrate introduces a novelplatform for integrating conventional chromatography, microfluidics andbiological assays (e.g Western blot analysis) with SERS, impartingchemical specificity to these techniques. Similar to microfluidicdevices, paper SERS based multiplexed detection of analytes from acomplex real-world sample can be a very powerful approach.Electrospinning of polymer fibers may also be a potential method forrealizing flexible SERS substrates. Electrospun polymer mats possess ahierarchical fibrous structure similar to that of paper, and the use ofdifferent types of polymers in electrospinning can bring better control(fiber diameter, alignment, surface chemistry) and multi-functionalityto the SERS design and applications.

All of the compositions and/or methods disclosed and claimed herein maybe made and/or executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of thisdisclosure have been described in terms of the embodiments includedherein, it will be apparent to those of ordinary skill in the art thatvariations may be applied to the compositions and/or methods and in thesteps or in the sequence of steps of the method described herein withoutdeparting from the concept, spirit, and scope of the disclosure. Allsuch similar substitutes and modifications apparent to those skilled inthe art are deemed to be within the spirit, scope, and concept of thedisclosure as defined by the appended claims.

It will be understood by those of skill in the art that information andsignals may be represented using any of a variety of differenttechnologies and techniques (e.g., data, instructions, commands,information, signals, bits, symbols, and chips may be represented byvoltages, currents, electromagnetic waves, magnetic fields or particles,optical fields or particles, or any combination thereof). Likewise, thevarious illustrative logical blocks, modules, circuits, and algorithmsteps described herein may be implemented as electronic hardware,computer software, or combinations of both, depending on the applicationand functionality. Moreover, the various logical blocks, modules, andcircuits described herein may be implemented or performed with a generalpurpose processor (e.g., microprocessor, conventional processor,controller, microcontroller, state machine or combination of computingdevices), a digital signal processor (“DSP”), an application specificintegrated circuit (“ASIC”), a field programmable gate array (“FPGA”) orother programmable logic device, discrete gate or transistor logic,discrete hardware components, or any combination thereof designed toperform the functions described herein. Similarly, steps of a method orprocess described herein may be embodied directly in hardware, in asoftware module executed by a processor, or in a combination of the two.A software module may reside in RAM memory, flash memory, ROM memory,EPROM memory, EEPROM memory, registers, hard disk, a removable disk, aCD-ROM, or any other form of storage medium known in the art. Althoughpreferred embodiments of the present disclosure have been described indetail, it will be understood by those skilled in the art that variousmodifications can be made therein without departing from the spirit andscope of the disclosure as set forth in the appended claims.

A controller, computing device, or computer, such as described herein,includes at least one or more processors or processing units and asystem memory. The controller typically also includes at least some formof computer readable media. By way of example and not limitation,computer readable media may include computer storage media andcommunication media. Computer storage media may include volatile andnonvolatile, removable and non-removable media implemented in any methodor technology that enables storage of information, such as computerreadable instructions, data structures, program modules, or other data.Communication media typically embody computer readable instructions,data structures, program modules, or other data in a modulated datasignal such as a carrier wave or other transport mechanism and includeany information delivery media. Those skilled in the art should befamiliar with the modulated data signal, which has one or more of itscharacteristics set or changed in such a manner as to encode informationin the signal. Combinations of any of the above are also included withinthe scope of computer readable media.

This written description uses examples to disclose the disclosure,including the best mode, and also to enable any person skilled in theart to practice the disclosure, including making and using any devicesor systems and performing any incorporated methods. The patentable scopeof the disclosure is defined by the claims, and may include otherexamples that occur to those skilled in the art. Such other examples areintended to be within the scope of the claims if they have structuralelements that do not differ from the literal language of the claims, orif they include equivalent structural elements with insubstantialdifferences from the literal language of the claims.

What is claimed is:
 1. A plasmonic biosensor comprising a nanostructurecore and functional monomers polymerized to the nanostructure core,wherein the polymer comprises at least one recognition cavity that issubstantially complementary to a target molecule.
 2. The plasmonicbiosensor of claim 1 wherein the nanostructure core comprises aplurality of nanostructure cores.
 3. The plasmonic biosensor of claim 1wherein the functional monomers comprise a silane, acrylamide, andcombinations thereof.
 4. The plasmonic biosensor of claim 3 wherein thesilane is selected from the group consisting of an organic silanemonomer, 3-aminopropyltrimethoxysilane, propyltrimethoxysilane,benzyltriethoxysilane, benzyldimethylchlorosilane,acetamidopropyltrimethoxysilane, and combinations thereof.
 5. Theplasmonic biosensor of claim 4 wherein the organic silane monomer isfunctionalized with a macromolecule selected from the group consistingof polyethylene glycol and albumin.
 6. The plasmonic biosensor of claim5 wherein the polyethylene glycol macromolecule is adsorbed to thefunctional monomers.
 7. The plasmonic biosensor of claim 1 furthercomprising a macromolecule selected from the group consisting ofpolyethylene glycol and albumin.
 8. A substrate comprising a plasmonicbiosensor comprising a nanostructure core and an agent that specificallybinds to a target molecule immobilized to the nanostructure core,wherein the plasmonic biosensor is adsorbed to a substrate.
 9. Thesubstrate of claim 8 wherein the substrate is selected from the groupconsisting of a paper and a fibrous mat.
 10. The substrate of claim 8wherein the nanostructure core is selected from the group consisting ofa gold nanostructure core, a silver nanostructure core, a coppernanostructure core, and combinations thereof.
 11. The substrate of claim8 further comprising a linker to immobilize the agent to thenanostructure core.
 12. A method of preparing a plasmonic biosensor, themethod comprising: synthesizing a nanostructure core; immobilizing atleast one template molecule on the surface of the nanostructure core toform a template molecule-nanostructure core structure; polymerizingfunctional monomers onto the template molecule-nanostructure corestructure; and removing the template molecule to form at least onerecognition cavity upon removal of the template molecule, wherein the atleast one recognition cavity is substantially complementary to a targetmolecule.
 13. The method of claim 12 wherein the functional monomerscomprise a silane, acrylamide and combinations thereof.
 14. The methodof claim 13 wherein the silane is selected from the group consisting ofan organic silane monomer, 3-aminopropyltrimethoxysilane,propyltrimethoxysilane, benzyltriethoxysilane,benzyldimethylchlorosilane, acetamidopropyltrimethoxysilane, andcombinations thereof.
 15. The method of claim 12 wherein the removingstep comprises exposing the template molecule-nanostructure corestructure to a reagent selected from the group consisting of oxalicacid, sodium dodecyl sulfate (SDS), and combinations thereof.
 16. Themethod of claim 12 further comprising adsorbing to the functionalmonomer a molecule selected from the group consisting of polyethyleneglycol and albumin prior to removing the template molecule.
 17. Themethod of claim 14 wherein the organic silane monomer is functionalizedwith a macromolecule selected from the group consisting of polyethyleneglycol and albumin.
 18. A method of preparing a substrate comprising aplasmonic biosensor, the method comprising: preparing a plasmonicbiosensor by a method comprising synthesizing a nanostructure core; andimmobilizing at least one agent to the nanostructure core, wherein theagent specifically binds to a target molecule; and adsorbing theplasmonic biosensor to a substrate.
 19. The method of claim 18 whereinthe substrate is selected from the group consisting of a paper and afibrous mat.
 20. The method of claim 18 wherein the nanostructure coreis selected from the group consisting of a gold nanostructure core, asilver nanostructure core, a copper nanostructure core, and combinationsthereof.
 21. The method of claim 18 wherein the at least one agent isimmobilized to the nanostructure core by a linker.